BACKGROUND:Carnosic acid,a bioactive compound found in rosemary,has been shown to reduce inflammation and reactive oxygen species(ROS).However,its mechanism of action in osteoclast differentiation remains unclear. OBJECTIVE:To investigate the effects of carnosic acid on osteoclast activation,ROS production,and mitochondrial function. METHODS:Primary bone marrow-derived macrophages from mice were extracted and cultured in vitro.Different concentrations of carnosic acid(0,10,15,20,25 and 30 μmol/L)were tested for their effects on bone marrow-derived macrophage proliferation and toxicity using the cell counting kit-8 cell viability assay to determine a safe concentration.Bone marrow-derived macrophages were cultured in graded concentrations and induced by receptor activator of nuclear factor-κB ligand for osteoclast differentiation for 5-7 days.The effects of carnosic acid on osteoclast differentiation and function were then observed through tartrate-resistant acid phosphatase staining,F-actin staining,H2DCFDA probe and mitochondrial ROS,and Mito-Tracker fluorescence detection.Western blot and RT-PCR assays were subsequently conducted to examine the effects of carnosic acid on the upstream and downstream proteins of the receptor activator of nuclear factor-κB ligand-induced MAPK signaling pathway. RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase staining and F-actin staining showed that carnosic acid dose-dependently inhibited in vitro osteoclast differentiation and actin ring formation in the cell cytoskeleton,with the highest inhibitory effect observed in the high concentration group(30 μmol/L).Carnosic acid exhibited the most significant inhibitory effect during the early stages(days 1-3)of osteoclast differentiation compared to other intervention periods.Fluorescence imaging using the H2DCFDA probe,mitochondrial ROS,and Mito-Tracker demonstrated that carnosic acid inhibited cellular and mitochondrial ROS production while reducing mitochondrial membrane potential,thereby influencing mitochondrial function.The results of western blot and RT-PCR revealed that carnosic acid could suppress the expression of NFATc1,CTSK,MMP9,and C-fos proteins associated with osteoclast differentiation,and downregulate the expression of NFATc1,Atp6vod2,ACP5,CTSK,and C-fos genes related to osteoclast differentiation.Furthermore,carnosic acid enhanced the expression of antioxidant enzyme proteins and reduced the generation of ROS during the process of osteoclast differentiation.Overall,carnosic acid exerts its inhibitory effects on osteoclast differentiation by inhibiting the phosphorylation modification of the P38/ERK/JNK protein and activating the MAPK signaling pathway in bone marrow-derived macrophages.

Objective To identify the enhancer landscape marked by histone H3K27ac modifications in diffuse-type gastric cancer(DGC)tissues,and to elucidate the epigenetic remodeling mechanisms by which active enhancers regulate cachexia-related genes.Methods Gastric mucosal tissue samples were collected from Department of Gastroenterology of Army Medical Center of PLA during January 2022 to March 2023,including 10 normal gastric mucosa tissues(Normal group),10 DGC tissues diagnosed with cachexia(DGC group),and 10 organoids derived from DGC tissues(Organoid group).Using H3K27ac chromatin targeting cleavage and tagmentation(CUT&Tag)technology,genomic modification regions were captured to screen specific active enhancers and their potential target genes in DGC tissues.CRISPR-dCas9 gene editing technology was used to intervene with the enhancers,and the expression of target genes was detected with Western blotting and qRT-PCR.Sixteen female SPF-grade BALB/c Nude mice(6～8 weeks old,weighing 18～21 g)were utilized to establish an orthotopic xenograft tumor model using the human diffuse-type gastric cancer cell line MKN45.Cachexia-related phenotypes were evaluated in 3 groups:normal group(n=4),silencing group(n=6),and control group(n=6).Results Significant differential enhancer regions were identified between DGC and normal gastric mucosa tissues.DGC tissues exhibited a marked increase in enhancer abundance(P<0.05)and signal intensity when compared with the normal counterparts.Integrated analysis of transcriptome data revealed that some of these active enhancers up-regulated the expression of GDF15,a cachexia-associated target gene in DGC.Targeted silencing of the active enhancer of GDF15 using CRISPR/dCas9-KRAB plasmid technology resulted in a significant reduction in GDF15 expression at both mRNA levels(P<0.05)and protein.Results from orthotopic transplantation experiments of DGC demonstrated that silencing of active enhancers alleviated the cachexia phenotype in nude mice(P<0.05).Conclusion DGC exhibits enhancer remodeling,which regulates the expression of the cachexia-associated gene GDF15,and thereby contributes to the pathogenesis and progression of cancer cachexia.

Objective:To explore the value of 3D reconstruction combined with watershed analysis in thoracoscopic anatomical partial lobectomy for peripheral early stage lung cancer.Methods:A retrospective cohort study was used to analyze the case data of 152 patients with early peripheral lung cancer who received targeted treatment in Hai′an People′s Hospital of Jiangsu Province from January 2022 to April 2023. All patients were treated with thoracoscopic anatomical partial lung resection. According to different preoperative analysis methods, they were divided into an observation group ( n=79) and a control group ( n=73). The observation group underwent three-dimensional reconstruction combined with venous drainage analysis, and the control group underwent routine analyze. The surgical indicators, pulmonary function indicators, C-reactive protein (CRP), interleukin-6 (IL-6) and complications were compared between the two groups. Normally distributed measurement data were expressed as mean±standard deviation ( ± s), independent samples t-test was used for comparison between groups, and paired-sample t-test was used for comparison within groups. Comparison of enumeration data between groups was performed using the chi-square test. Results:The operation time, postoperative drainage time, postoperative hospital stay, and intraoperative blood loss in the observation group were (76.57±11.94) min, (2.15±0.24) d, (4.32±0.75) d, and (27.63±3.54) mL respectively. The control group was (112.64±13.67) min, (3.76±0.31) d, (5.16±0.81) d, (53.26±4.61) mL, respectively. The observation group was smaller than the control group, and the differences between the two groups were statistically significant ( P<0.05). At discharge, the observation group had maximum ventilation per minute as a percentage of predicted value (MVV% pred), forced expiratory volume in 1 second as a percentage of predicted value (FEV1% pred), and forced vital capacity as a percentage of predicted value (FVC% pred) were (83.33±7.51)%, (85.22±7.44)%, (84.16±7.26)%, and those of the control group were (77.19±7.46)%, (76.47±7.36)%, and (79.38±7.20)%, respectively. The observation group was greater than that of the control group, and the differences between the two groups were statistically significant ( P<0.05). The CRP and IL-6 in the observation group were(47.53±6.34) mg/L, (32.21±5.14) mg/L, (144.53±20.84) ng/L, (180.82±21.17) ng/L, respectively on the 1st and 3rd days after surgery, while those in the control group were (57.84±6.71) mg/L, (38.77±5.23)mg/L, (155.14±22.83) ng/L, (200.97±25.52) ng/L, the observation group was lower than that of the control group, and the difference was statistically significant ( P<0.05). The incidence rate of complications in the observation group was 5.06%, and that in the control group was 10.96%, There was no statistically significant difference between the two groups ( P>0.05). Conclusion:Preoperative 3D reconstruction combined with venous drainage analysis can better promote the recovery of patients with early peripheral lung cancer and retain more lung function after thoracoscopic anatomical partial pulmonary resection.

Objective To investigate the relationship between serum L-carnitine and its related metabolites[trimethylamine(TMA)and trimethylamine N-oxide(TMAO)]levels and gestational diabetes mellitus(GDM)in the second trimester of pregnant women in Shanghai.Methods A case-control study was conducted in 280 pregnant women between 18 and 23 weeks of gestation from January 2018 to January 2021.Among them,134 cases of GDM were the case group(GDM),and 146 cases with normal blood glucose(BG)were the control group(Con).Serum L-carnitine,TMA and TMAO levels were quantified by ultra high performance liquid chromatography-mass spectrometry.Logistic regression analysis,stratified analysis and linear regression were used to explore the relationship between L-carnitine,TMA and TMAO levels and GDM and glucolipid metabolism.Results Serum L-carnitinelevelwas significantly lower in GDM group than that in Con group(P<0.01).After adjusting for confounders,logistic regression showed a 70%reduction in the risk of GDM in the group with highest tertile of L-carnitine compared with the group with lowest tertile(OR 0.30,95%CI 0.15～0.63).The risk of GDM decreased by 14%for each 1 μmol/L increase in serum L-carnitine(OR 0.86,95%CI 0.80～0.93).Serum L-carnitine was negatively correlated with 1 hPG(r=-0.21,P<0.01)and 2 hPG(r=-0.15,P<0.05),respectively,TMA was negatively correlated with 2 hPG(r=-0.21,P<0.01).Conclusions Higher serum L-carnitine level may be negatively associated with GDM.Serum L-carnitine and TMA levels were negatively correlated with blood glucose levels.

Objective To explore the factors influencing the occurrence of cardiac complications after endoscopic surgery in patients with non-small cell lung cancer(NSCLC)and construct the prediction model.Methods A total of 312 patients with non-small cell lung cancer who were diagnosed and underwent thoracoscopic treatment in our hospital from January 2020 to January 2023 were selected as the objects of this study,and were divided into cardiac complication group and non-cardiac complication group according to whether cardiac complications occurred after surgery.The clinical data of patients in each group were analyzed,and the factors affecting the occurrence of cardiac complications in patients with non-small cell lung cancer after endoscopic surgery were discussed by multivariate Logistic regression analysis.A full-variable risk prediction model for the occurrence of cardiac complications was constructed,and the predictive model was verified by receiver operating characteristic curve(ROC)analysis.Results The incidence of postoperative cardiac complications in 312 patients with non-small cell lung cancer was 24.04％.Univariate analysis showed that Age,smoking history,smoking history,comorbidities,intraoperative blood loss,operative time,systolic blood pressure,FEV1,FEV1％,FPG,HbA1c and urinary microalbumin were correlated with the incidence of cardiac complications in patients with non-small cell lung cancer after endoscopic surgery(P＜0.05).Multivariate Logistic stepwise regression analysis showed:age,comorbidities,operation time and HbA1c were independent risk factors for postoperative cardiac complications in patients with non-small cell lung cancer(P＜0.05).A risk prediction model for cardiac complications was established based on multiple factors,that is,P=1/(1+e-y),y=-3.114+0.881 × age+0.754 × complications+1.285 × operation time+1.185 × HbA1c,and the ROC curve diagnosis showed that the risk prediction model predicted the occurrence of cardiac complications with an AUC of 0.847,95％CI of 0.769-0.925,sensitivity of 82.5％,specificity of 83.2％.Conclusion Age,complications,operation time,and HbA1c are all independent risk factors affecting the occurrence of cardiac complications in patients with non-small cell lung cancer after endoscopic surgery,and the risk prediction model constructed by the above factors has a good predictive value for the occurrence of cardiac complications.

Objective:To study the mechanism of Herba Hedyotidis against liver fibrosis based on network pharmacology. Methods:Based on TCMSP database and Uniprot database, the effective components and target genes of Herba Hedyotidis were screened. Target genes of liver fibrosis were screened by GeneCards and OMIM database, and the "disease-component-target" network map was constructed by Cytoscape 3.8.2 software. Protein interaction network was constructed by STRING database, and the Cytoscape 3.8.2 software was used to screen the core target out. The core targets were analyzed by gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Experimental verification was performed to the analysis results. A hepatic fibrosis model was established by intraperitioneal imjection of 40% carbon tetrachloride oil solution in rats that were then divided into the model control group and the Herba Hedyotidis group by randomized number table table, with 10 rats in each group. Ten normal rats were used as the normal control group. The Herba Hedyotidis group were injected 2.7 g/kg herb aqueous extract by intragastric administration, once a day, for 4 weeks; and the normal and model control group were given the same volume distilled water for gavage. The serum GPT, GOT, Alb and liver pathologic changes were observed. The serum expressions of IL-6, IL-1β and TGF-β1 were detected by ELISA. The expressions of PI3K, Akt, HIF-1α and VEGF were detected by Western blot. Results:5 effective components and 118 targets of Herba Hedyotidis in the treatment of hepatic fibrosis were obtained. Stigmasterol, β-sitosterol and quercetin were the most effective components with high moderate value. The moderate targets were VEGF, EGFR, HIF-1α and IL-6. The core genes of PPI network were HIF-1α, IL-6, etc. GO enrichment analysis showed that RNA transcription, protein binding and other processes may be affected. KEGG pathway enrichment analysis showed that significant enrichment pathways were cancer pathway, hepatitis B pathway, PI3K/Akt, HIF pathway and so on. Animal experimental results showed that compared with model group, liver histopathology was improved significantly, the content of GPT, GOT, IL-6, IL-1β and TGF-β1 decreased ( P<0.01), the content of Alb increased ( P<0.01), and the protein expressions of PI3K, Akt, HIF-1α and VEGF in liver tissue were down-regulated ( P<0.01). Conclusion:The Herba Hedyotidis exerts functions of anti-hepatic fibrosis through acting on the targets of VEGF, EGFR, HIF-1α and IL-6, regulating the PI3K/Akt, HIF-1 pathways, and has anti-inflammatory, anti-angiogenesis, anti-tumor and other biological functions.

Background Ostrich acellular corneal stroma possesses a similar constitution to human corneal stroma,so it is expected to become one of ideal biological corneal carriers.Objective This study was to investigate the immunogenicity of acellular stroma carrier of ostrich cornea and offer the information for the development of industrialization and clinical use of acellular stroma carrier of ostrich cornea.Methods Twenty fresh ostrich eyeballs and 20 porcine eyeballs were collected.Acellular corneal stroma carriers of ostriches and pigs were prepared using low temperature freezing joint enzyme digestion method and desiccant dehydration method and sterilized by cobalt-60 irradiation.The corneal stroma carriers were preserved using drying and dehydration method.Forty-five male BALB/c mice were randomly divided into the sham operation group,ostrich acellular corneal stroma group and porcine acellular corneal stroma group.Acellular corneal stroma carriers of ostriches and pigs(wet weight after rewatering was 10 mg/piece) were subcutaneously implanted to the back of BALB/c mice,respectively.Wound healing and inflammatory response on the operative site were observed,and phenotype and activating rate of CD4+,CD8+ and CD25+in peripheral blood of mice were dynamically detected 7,14 and 28 days after ectopically implantation of heterogeneous corneal stroma by immunofluorescence labeling and flow cytometry analysis.Results No swelling and exudation were seen in the skin of operative site of the mice with a good healing of wound after surgery.There were no significant differences in the activating rates of CD4+,CD8+ and CD25+ cells in the peripheral blood of mice among the sham operation group,the porcine acellular corneal stroma group and ostrich acellular corneal stroma group in the three time points after surgery(CD4+:F=0.74,P=0.50;F=0.39,P=0.05;F=3.46,P=0.58.CD8+:F=1.75,P=0.21 ;F=1.14,P=0.35;F=0.78,P=0.48.CD25+:F=0.52,P=0.61 ;F=3.53,P=0.62;F=2.42,P=0.13).Conclusions The ostrich acellular heterogeneous corneal stroma carrier possesses low immunogenicity.It is inferred that ostrich acellular corneal stroma carrier can be used in heterogeneous corneal transplantation.

Background Fungal corneal ulcer is a visual-threatening eye disease,and drug therapy has a limiting efficacy.Corneal transplantation or eye enucleation sometimes is necessary to the severe patients.Corneal collagen cross-linking (CXL) is an effective method for some corneal diseases,but the study on CXL for fungal corneal ulcer is lack.Objective This study was to evaluate the clinical effectiveness and safety CXL for fungal corneal ulcer.Methods Fifteen 8-week-old healthy New Zealand white rabbits were used in this study and other 5 rabbits served as normal controls.Fungal corneal ulcer models were established in the right eyes of other 10 rabbits by infecting sickle bacteria liquid after corneal scratching and removing corneal epithelium,then decellularized ostrich corneal patch covered the defected cornea.The models were randomly divided into the non-treatment group and the CXL treatment group.Corneal lesions were examined under the slit lamp microscope every day,and cornea was pictured by laser scanning confocal microscope on the 3rd,7th,14th,21st and 28th day individually after CXL.All rabbits were sacrificed and corneal tissues were obtained 4 weeks after treatment,and the collagen fiber diameter and fibrocytes were observed under the scanning electron microscope.Results Fungal corneal ulcer models were successfully established by corneal scratching and decellularized ostrich cornea covering.The gray ulcer lesions and hypbae like bean pod were seen by slit lamp microscope and laser scanning confocal microscope 3 days after modeling.Corneal ulcer deepened and expanded 1 week later,and there were a large number of spore and hyphae criss-crossing as short rod in shallow stroma.Inflammatory cells were observed in corneal endothelial cells and ocular anterior chamber.In the CXL treatment group,the range of corneal epithelial deficiency was less than that in the nontreatment group on the 3rd,7th,14th,and 21st (all at P＜ 0.05).The diameters of collagen fibers were (24.6± 1.8) nm,(24.9 ± 1.9) nm and (43.0 ± 7.4) nm in the normal control group,non-treatment group and CXL treatment group,showing a significant difference among the 3 groups (F =27.05,P =0.00),and the collagen diameters were thicker in the CXL treatment group than those in the normal control group and non-treatment group (t =5.40,-5.30,both at P＜0.05),and fibrocytes were seen among the collagen fibers.No significant difference was found in the collagen diameters between the non-treatment group and normal control group,and the fibrocytes were less in the non-treatment group.Conclusions CXL therapy can treat fungal corneal ulcer by enhancing collagen,promoting fibrocytes proliferation,suppressing fungus and inflammatory response and accelerating tissue repair.

Objective To explore the incidence and factors of paranasal sinusitis among nasopharyngeal carcinoma (NPC) patients after radiotherapy. Methods Retrospectively evaluated the clinical data of 144 NPC patients without paranasal sinusitis before radiotherapy, including 82 cases in T1/T2 stage and 62 cases in T3/T4 stage treated in the First Affiliated Hospital of Bengbu Medical College from 2000 to 2005. MRI images before and after radiotherapy were compared. The incidence and factors of paranasal sinusitis were analyzed. There were 58 cases of nasal invasion. Nasopharyngeal carcinoma was given at face-neck joint portal with 6 MV X-ray fractionated irradiation 68-78 Gy during 6-8 weeks. The number of patients who received radiotherapy with less than or equal to 70 Gy, more than 70 Gy radiation doses were 89 and 55, respectively. Cervical part were treated with high-energy electron beam, patients with positive neck lymph nodes and with negative neck lymph nodes received 64-74 Gy doses during 6-8 weeks and 50-54 Gy during 4-5 weeks, respectively. Results Among the 144 NPC patients 86.8%(125/144) developed paranasal sinusitis after radiotherapy, the incidence rates of paranasal sinusitis (IRPS) was higher among stage T3 + T4 patients than that among stage T1 + T2 patients (94% vs 82% ,x2=4.32, P ＜0.05). Among patients who were given radiotherapy with more than 70 Gy,less than or equal to 70 Gy radiation doses on the nasopharynx, the IRPS were 95% and 82.0% (x2 = 4.65, P ＜ 0.05 ). The IRPS in patients with nasal cavity infringement was higher than that in others (95% vs. 81% , x2 = 5.46,P ＜0.05). The IRPS at 3, 6, 12 months, and more than 1 year after radiotherapy were 13.6% ,31.2% ,48.8% and 6.4%, respectively (x2 = 70.48, P ＜ 0.001 ). Conclusions The incidence of paranasal sinusitis in NPC patients after radiotherapy was very high, and reached a peak in one year. It was influeneed by invasion of nasal cavity or not, the dose of radiotherapy and T stage.

Objective To study the hemodynamic characteristics of ductus venosus,and its relationship to inflammatory cytokines in ovine fetus with systemic inflammatory response syndrome.Methods Ten near-term fetal sheep radomly divided into two groups.Five of them were in experimental group,and five of them were in control group.All of the animals underwent abdominal cordocentesis guided by ultrasound at the term of 120-130 days,with a LPS injection at a dosage of 10 μg/kg of fetal weight in experimental group,and the same quantity of 0.9% NaCl solution in control.Doppler echocardiography were performed to determine hemodynamics changes of ductus venosus at 0.5 hour before the LPS injection,and at 1 hour,3 hours,6 hours after LPS injection.Meanwhile,fetal umbilical vein blood was sampled for ELISA essay of serum TNF-α and IL-6 at each of the above time points.Results In experimental group,the ductus venosus waveform PI values(DVPI),maximum velocity during cardiac ventricle systole(S)and during cardiac ventricle diastole(D),ductus venosus index(DVI),ductus venosus blood flow(DVQ),the ratio of S and ductus venous maximum velocity during atrial contracton(S/A),(S-A)/D increased with the time after LPS injection,and still significantly increased as compared with the control animals(all P＜0.05).A decreased with the time after LPS injection,and still significantly changed as compared with the controls(P＜0.05).And the ductus venosus diameter(DVD)had not obvious change(P＞0.05).There were significantly positive correlations between DVPI and TNF-α,IL-6(all P＜0.05),negative corelations between A and TNF-α,IL-6(all P＜0.05),and positive correlations between S wave,DVI,DVQ,D wave,S/A,(S-A)/D and IL-6(all P＜0.05),but not obvious relationships between S wave,DVI,DVQ,D wave,S/A,(S-A)/D and TNF-a(all P＞0.05).Condusions DVPI and A might be easy and useful quantitative parameters in the evaluation of fetal systemic inflammatory respome syndrome with LPS injection.